Walter BE et al. (2008), Psf2 plays important roles in normal eye develo...

XB-ART-37805

Mol Vis 2008 May 19;14:906-21.

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Psf2 plays important roles in normal eye development in Xenopus laevis.

Walter BE , Perry KJ , Fukui L , Malloch EL , Wever J , Henry JJ .

Abstract
PURPOSE: Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a "sliding clamp." Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis. METHODS: A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens. RESULTS: Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay. CONCLUSIONS: The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA).

PubMed ID: 18509549
PMC ID: PMC2391082

Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: gins2 gins4
Antibodies: Lens Ab1
Morpholinos: gins2 MO1

Article Images: [+] show captions

	Figure 2. Summary of the effects of Psf2MO injection on eye development. Shading depicted in the key indicates categories of normal, minor, and severe eye defect phenotypes (see text for explicit definitions). A: Uninjected control embryos and those injected with the control morpholino (CONMO) exhibited minimal affects. B: Embryos injected with lissamine-tagged Psf2MO exhibit dose dependant eye defects. Note the increasing number and severity of eye defects with increasing doses of Psf2MO. C: Embryos co-injected with Psf2MO and altPsf2 RNA exhibit a dose dependent reduction in eye defects. Error bars indicate standard error.
	Figure 3. Results of Psf2 morpholino knockdown and RNA rescue experiments. Dorsal is toward the top in each figure. A-F: Typical eye defects observed following unilateral injection of lissamine-tagged Psf2MO into single blastomeres at the two-cell stage (equivalent to 2.25 ng/cell at the eight-cell stage) are shown. A: Normal control (uninjected, CON) side is shown. This side is the normal part the embryo shown in B-C. B: Minor eye defect is observed on the Psf2MO-injected side (opposite that shown in A) as indicated by arrow. Note the slight decrease in retinal pigmentation in the ventral region and the reduced size of the optic cup compared to that shown in A. C: Corresponding fluorescence image to that shown in B, the image shows the distribution of the lissamine-tagged morpholino (LIS). D: Normal control, uninjected side is shown. This side is the normal part of the embryo shown in E-F. E: A severe eye defect phenotype is observed following Psf2MO injection. Note the smaller size of the eye and severe reduction in the ventral region of the optic cup, denoted by the arrow in E. F: The corresponding fluorescence image shows the distribution of the lissamine-tagged morpholino for the embryo shown in E. G-I: The typical result observed following the co-injection of 2.25 ng Psf2 morpholino and 1000 pg synthetic altPsf2 RNA is shown. G: The normal control, uninjected side of the embryo shown in H-I is displayed. H: There was normal morphological development following the co-injection of Psf2MO (equivalent to 2.25 ng/blastomere at the eight-cell stage) and 1000 pg rescue RNA (altPsf2 RNA). I: The corresponding fluorescence image to that shown in H shows distribution of the lissamine-tagged morpholino. J-L: Typical severe eye defect is observed following the unilateral injection of lissamine-tagged Psf2MO into single blastomeres at the two-cell and four-cell stages (equivalent to 4.5 ng/blastomere at the eight-cell stage). J: The normal control, uninjected side of the embryo shown in K-L is pictured. K: Typical severe eye defect phenotype is observed following Psf2MO injection. Note the smaller size of the eye and severe reduction in the ventral region of the optic cup, denoted by the arrow in K. L: The corresponding fluorescence image shows the distribution of the lissamine-tagged morpholino for the embryo shown in K. M-O: The typical result is observed following the co-injection of 4.5 ng Psf2 morpholino and 1000 pg synthetic altPsf2 RNA. M: The normal control, uninjected side of the embryo shown in N-O is displayed. N: Normal morphological development is observed following the co-injection of Psf2MO (equivalent to 4.5 ng/blastomere at the eight-cell stage) and 1000 pg rescue RNA (altPsf2 RNA). O: The corresponding fluorescence image to that shown in N shows the distribution of the lissamine-tagged morpholino. P-R: Typical severe eye defect is observed following unilateral injection of lissamine-tagged Psf2MO into single blastomeres at the two-cell and four-cell stages (equivalent to 6 ng/blastomere at the eight-cell stage). P: The normal control, uninjected side of the embryo shown in Q and R is shown. Q: The typical severe eye defect phenotype is displayed following Psf2MO injection. Note the severe reduction in the ventral region of the optic cup, denoted by the arrow in Q, and the overall lack of retinal pigmentation. R: The corresponding fluorescence image shows the distribution of the lissamine-tagged morpholino for the embryo shown in Q. S-U: The typical result is observed following co-injection of 6 ng Psf2 morpholino and 1000 pg synthetic altPsf2 RNA. S: The normal control, uninjected side of the embryo shown in T-U is displayed. T: There is normal morphological development following the co-injection of Psf2MO (equivalent to 6 ng/blastomere at the eight-cell stage) and 1000 pg rescue RNA (altPsf2 RNA). U: The corresponding fluorescence image to that shown in S shows the distribution of the lissamine-tagged morpholino. The scale bar in U is equal to 450 µm.
	Figure 4. Transverse sections of specimens demonstrating severe eye defects following Psf2MO-mediated knockdown. Dorsal is toward the top in each figure. A: Image montage of a typical specimen shows the Psf2 morpholino-injected side on the left and the internal control, uninjected side on the right. This image was viewed with differential interference contrast (DIC). The red color in A shows overlain distribution of rhodamine fluorescence of secondary antibodies revealing immunoreactive lens crystallin proteins within both lenses. The cornea epithelium overlying the eye on the left is thicker compared to the uninjected side on the right, characteristic of undifferentiated embryonic ectoderm. Note that the lens and retina of the morpholino-affected eye are smaller and not as fully differentiated. The forebrain is also smaller and less differentiated on the left, Psf2MO-injected side. B and C are the higher magnification views of the left and right eyes shown in A, respectively. D-E: The left and right sides of the head of a second case stained with Hematoxylin/Eosin are shown. F and G are higher magnification views of the eyes shown in D and E, respectively. Note that the forebrain and retina are malformed on the left, Psf2MO-injected side shown in D and F compared to the control side shown in E and G. On the Psf2MO-injected side (D, F), only a small lens vesicle possessing a central lumen has formed. This lens vesicle exhibits some polarization and evidence of elongating primary fiber cells. Also note the retarded differentiation of the cornea in D and F compared to the respective control cornea shown in E and G. H-I: More posterior sections show the reduction in hindbrain size and the absence of the otic vesicle on the Psf2MO-injected side (H) compared to the normal pattern of development seen on the control, uninjected side (I). cn, cornea epithelium; fb, forebrain; fg, foregut; hb, hindbrain; le, lens epithelium; lf, lens fiber cells; ln, lens; lv, lens vesicle; on, optic nerve; ov, otic vesicle; rpe, retinal pigmented epithelium; rt, neural retina. The scale bar in I is equal to 160 µm in A-B, 85 µm in B-C, 170 µm in D-E, 100 µm in F-G, and 190 µm in H-I.
	Figure 5. Diagrams illustrating the tissue transplantation experiment performed to localize Psf2 function in the eye. This experiment involves reciprocal transplantation of stage 14 presumptive lens ectoderm (ple) between uninjected embryos (upper example) and morpholino (MO)-injected embryos (lower example). Single blastomeres were injected with morpholino at the four-cell stage as shown. Green color shows the distribution of the co-injected morpholino and GFP RNA tracer. See text for further details.
	Figure 6. Summary of the data obtained from the reciprocal tissue transplantation experiments diagramed in Figure 5. See also the text and Figure 7. aGreen color denotes the presence of the morpholino. bData from Henry and Grainger is shown [18]. HRP stands for horseradish peroxidase. The exact lens sizes were not reported in that study but were less than the full size. cSee Methods for details on these measurements.
	Figure 7. Examples of the results observed from reciprocal presumptive lens ectoderm transplants between control and Psf2MO-injected embryos. Dorsal is toward the top in each figure. Arrowheads point to eyes in the whole mounts shown in A-C and H-J. A-G: This example shows the typical result that is observed when the presumptive lens ectoderm from a control embryo is transplanted to the lens-forming region of a Psf2MO-injected host (see Figure 5, the text, and Figure 6). A: The view of the control (“CON”), unoperated side of the larva is shown. B: The view of the operated side that received the transplanted PLE (“TRANSPLANT”) shows abnormal development of the retina and lens. C: The whole mount fluorescence image corresponds to that shown in B, which reveals the location of the transplanted ectoderm via distribution of GFP expressed in the donor tissue (“GFP”). D and E: High magnification DIC images of transverse sections through the control side (D) and the operated side that received the transplanted tissue (E) are displayed. F and G: Corresponding immunofluorescence images show anti-lens antibody staining of the sections shown in D and E, respectively. Note formation of an abnormal retina and small lens body in E and G. H-N: This example shows the typical result that is observed when the presumptive lens ectoderm from a Psf2MO-injected embryo is transplanted to the lens-forming region of a control host (see Figure 5, the text, and Figure 6). H: The view of the control (“CON”), unoperated side of the larva is shown. I: The view of the operated side that received the transplanted PLE (“TRANSPLANT”) shows smaller overall size of the eye. J: The whole mount fluorescence image corresponds to that shown in I, which reveals the location of the transplanted ectoderm via distribution of GFP expressed in the donor tissue (“GFP”). K-I: High magnification DIC images of transverse sections through the unoperated, control side (K), and the side that received the PLE transplant derived from the Psf2MO-injected embryo (L), are shown. M and N display the corresponding immunofluorescence images showing anti-lens antibody staining of the sections presented in K and L, respectively. Note that the retina and lens formed on the operated side (L and N), although smaller compared to the unoperated side (K and M), exhibit fairly normal morphology. Labels are the same as those used in Figure 4. lb stands for lens body. The scale bar in N is equal to 450 µm in A-C and H-J and 80 µm in D-G and K-N.
	Figure 8. Effects of Psf2MO and CONMO injections on cell proliferation in the neural retina and lens. A-H: Transverse sections of eyes in Psf2MO-injected and CONMO-injected embryos show corresponding pairs of differential interference contrast and fluorescence micrographs. Fluorescence micrographs in B, D, F, and H show distribution of proliferating cells labeled with anti-phospho-histone H3 S10P antibody (green). White arrowheads point to examples of these labeled cells within the retina. A and B: The normal, control eye derived from the uninjected side of one example is displayed. C and D: Opposite, defective eye derived from the Psf2MO-injected side of the same embryo shown in A-B is displayed in these panels. E and F: The normal, control eye derived from the uninjected side of another embryo is shown. G and H: Opposite, normal-appearing eye derived from the CONMO-injected side of the same embryo shown in E-F is displayed in these panels. I: Graphical depiction of the levels of cell proliferation in the neural retina and the lens is shown. Bars represent the mean fraction of histone H3 S10P labeled cells (depicted as a percentage along the y-axis) while the different tissues and conditions examined are depicted along the x-axis, as indicated. Error bars representing the standard deviation are also shown. See Methods for further details explaining the preparation of this data. Labels are the same as those used in Figure 4. Scale bar in H equals 100 µm.
	Figure 9. Effects of Psf2MO and CONMO injections on the level of apoptosis. A-B,D-E: Whole mount examples show gross distribution of apoptotic cells (containing blue colored NBT-BCIP precipitate). These whole mount embryos have been cleared with BABB. A and B: These views of an embryo show sides derived from Psf2MO-injected and uninjected blastomeres, respectively. C: Transverse section through the head of a Psf2MO-injected embryo is shown. The white dashed line separates the side containing tissues derived from the Psf2MO-injected blastomere (on the left side of the figure) from those derived from the uninjected blastomere (on the right side of the figure). D and E: These views of an embryo show sides derived from CONMO-injected and uninjected blastomeres, respectively. F: Transverse section through the head of a CONMO-injected embryo is shown. The white dashed line separates the side containing tissues derived from the CONMO-injected blastomere (on the left side of the figure) from those derived from the uninjected blastomere (on the right side of the figure). Note the increased level of apoptosis in head tissues derived from Psf2MO-injected cells, especially in the forebrain and neural retina (e.g., compare A versus D and C versus F). Black arrowheads point to examples of labeled apoptotic cells within the retina and brain. G: A graphical depiction of the levels of apoptosis in the neural retina and the lens is displayed. Bars represent the mean fraction of apoptotic cells (depicted as a percentage along the y-axis) while the different tissues and conditions examined are depicted along the x-axis, as indicated. Error bars representing the standard deviation are also shown. See Methods for further details explaining the preparation of this data. Labels are the same as those used in Figure 1 and Figure 4. Scale bar in F equals 600 µm for A-B and D-E and 110 µm for C and F.
	Figure 1. Embryonic expression of Psf2 [renamed gins2]. A: An example of a whole mount in situ hybridization pattern showing localization of Psf2 in specific embryonic tissues (stage 33) is shown. Note the expression in the brain (labeled as cns), the retina and lens of the eye (labeled as eye), mesoderm of the pharyngeal arches (labeled as pa), and in stripes representing a reiterated subset of the paraxial (somitic) mesoderm (labeled as pm). B: RT–PCR analysis of Xenopus laevis Psf2 at different stages of embryogenesis, as noted (all stages follow those of [15]). A portion of 1 kb ladder was run for reference (labeled as 1kb). The 1018 bp and 506 bp bands are labeled. Expected Psf2 PCR product is 577 bp. For simplicity, positive and negative control lanes are not shown here.

References [+] :

Breitman, Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage. 1989, Pubmed